    (1) Reviews: a) Tam, J. P.; Xu, J.; Eom, K. D. Biopolymers (Peptide Sci.) 2001, 60, 194-205; b) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 91-118; c) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem. Int. Ed. 2008, 47, 10030-10074; d) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338-351.    (2) Efforts to overcome this requirement include: a) Tam, J. P.; Yu, Q. Biopolymers 1998, 46, 319-327; b) Offer, J.; Boddy, C. N. C.; Dawson, P. E. J. Am. Chem. Soc. 2002, 124, 4642-4646; c) Wu, B.; Chen, J.; Warren, J. D.; Chen, G; Hua, Z.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2006, 45, 4116-4125; d) Botti, P.; Tchertchian, S. WO/2006/133962; e) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064-10065; f) Payne, R. J.; Fichet, S.; Greenberg, W. A.; Wong, C.-H. Angew. Chem. Mt Ed. 2008, 47, 4411-4415; g) Okamoto, R.; Kajihara, Y. Angew. Chem. Int. Ed. 2008, 47, 5402-5406; h) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem. Int. Ed. 2008, 47, 6807-6810; i) Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2008, 47, 8521-8524; j) Bennett, C. S.; Dean, S. M.; Payne, R. J.; Ficht, S.; Brik, A.; Wong, C.-H. J. Am. Chem. Soc. 2008, 130, 11945-11952; k) Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X.-W.; Liu, C.-F. J. Am. Chem. Soc. 2009, 131, 13592-13593; l) Harpaz, Z.; Siman, P.; Kumar, K. S. A.; Brik, A. ChemBioChem 2010, 11, 1232-1235; m) Chen, J.; Wang, P; Zhu, J.; Wan, Q.; Danishefsky, S. J. Tetrahedron 2010, 66, 2277-2283; n) Shang, S.; Tan, Z.; Dong, S.; Danishefsky, S. J. J. Am. Chem. Soc. 2011, 133, 10784-10786.    (3) Danishefsky has reported the HOBt-mediated oxidative coupling of peptide thioacids and free N-terminal peptides. This method is not compatible with unprotected sidechain amines. Wang. P.; Danishefsky, S. J. J. Am. Chem. Soc. 2010, 132, 17045-17051.    (4) Okawa, K.; Nakajima, K. Biopolymers 1981, 20, 1811-1821.    (5) Korn, A.; Rudolph-Böhner, S.; Moroder, L. Tetrahedron 1994, 50, 1717-1730.    (6) Galonic, D. P.; Ide, N. D.; van der Donk, W. A.; Gin, D. Y. J. Am. Chem. Soc. 2005, 127, 7359-7369.    (7) Shao, H.; Jiang, X.; Gantzel, P.; Goodman, M. Chemistry & Biology 1994, 1, 231-234.    (8) The C2-selective opening of NH aziridine-2-carbonyl-terminated peptides (formed in situ from β-bromoalanylpeptides) by peptide thioacids to give a β-peptide linkage (after S- to N-acyl transfer) was originally observed by Tam et al.: Tam, J. P.; Lu, Y. A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. USA 1995, 92, 12485-12489.    (9) Recently, a convergent synthesis of protected peptidomimetics via the coupling of protected peptide thioacids and protected 2-aziridinylmethylpeptides was reported: Assem, N.; Natarajan, A.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132, 10986-10987.    (10) Ag(I) ion is known to promote the oxidative coupling of thioacids and primary amines: a) Schwabacher, A. W.; Bychowski, R. A. Tetrahedron Lett. 1992, 33, 21-24; b) Blake, J. Int. J. Peptide Protein Res. 1981, 17, 273-274; c) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. USA 1981, 78, 4055-4058.    (11) K3Fe(CN)6, is known to promote the N-acylation of primary amines via dithioacids: Liu, R.; Orgel, L. E. Nature 1997, 389, 52-54.    (12) Thioacid 8 was prepared from commercially available Ac-Phe-OH (1. NHS, DCC, DCM, rt, 4 h; 2. NaHS, MeOH, 63% yield) using a known method: Goldstein, A. S.; Gelb, M. H. Tetrahedron Lett. 2000, 41, 2797-2800.    (13) The structure of 14 was confirmed through comparison with an authentic sample prepared using standard peptide coupling protocols.    (14) The Fmoc protecting group was retained in this example to facilitate quantitative determination of the epimer ratio.    (15) Peptide thioacids 17, 19, and 21 were prepared by deprotection (TFA, DCM, Et3SiH, 0° C.) of their STmb thioester precursors in 73, 53, and 45% yields.    (16) MS analysis of this coupling reaction indicated predominant formation of a disulfide corresponding to intermediate 12, which implies that the free thiol may be undergoing an in situ protection. Reductive disulfide cleavage likely occurs during the workup with aqueous NaSH, which can act as a reducing agent. Minor products emanating from perthioester intermediates were also detected. See: Liu, C. F.; Rao, C.; Tam, J. P. Tetrahedron Lett., 1996, 37, 933-936.    (17) The aziridine-containing tripeptide 24 was prepared from the union of Tr-Azy(Me)-OH and H-Phe-Gly-NH2 (HATU, DIEA, DMF, rt, 48% yield) followed by deprotection (TFA, (1:1) CHCl3-MeOH, 0° C., 61% yield).    (18) A protocol for ligation at Thr via chemical ligation of a γ-thiol-substituted N-terminal Thr peptide followed by post-ligation desulfurization was recently reported. See reference (2m).I. Experimental ProceduresIa. General Considerations. Reagent grade solvents were used for extraction and flash chromatography. All reagents and solvents were purchased from commercial sources and were used without further purification unless otherwise noted. The progress of reactions was monitored by analytical thin layer chromatography (TLC, silica gel F-254 plates) or analytical HPLC (see below). TLC plates were visualized first with UV illumination (254 nm) followed by charring using either ninhydrin stain (0.3% ninhydrin (w/v) in 97:3 EtOH/AcOH) or a modification of Hanessian's stain (10 g ammonium molybdate ((NH4)6Mo7O24.4H2O) and 5 g cerium sulfate (Ce(SO4)2) in 1 L 10% aq. H2SO4). Aqueous NaSH was prepared fresh daily by dissolving ˜50 mg NaSH hydrate in 1-2 mL of water. Flash column chromatography was performed on flash grade (230-400 mesh) silica gel. The solvent compositions reported for all chromatographic separations are on a volume/volume (v/v) basis. Solvent removal under reduced pressure was performed by rotary evaporation (pressure ˜16 mm Hg, bath temperature 25-30° C.) followed by pumping under high vacuum until the container reached a constant mass. High performance liquid chromatography (HPLC) was carried out using an X-Bridge C18 (3×250 mm column) for analytical separations and X-Bridge C18 (19×150 mm) column for semipreparative purifications. HPLC Eluent B was a solution of 0.1% TFA in MeCN and Eluent A was a 0.1% aqueous solution. HPLC analysis was monitored using dual channel UV detection at 254 and 215 nm. All peptide products purified by preparative HPLC were isolated by removing the MeCN and free TFA by rotary evaporation and the remaining water by lyophilization. Melting points are uncorrected. Optical rotations were recorded at room temperature at the sodium D line (589 nm). 1H NMR spectra were recorded at ambient temperature, at 300 or 600 MHz, and are reported in parts per million (ppm) on the δ scale relative to tetramethylsilane (δ0.00). 13C NMR spectra were recorded at 75.5 or 150.8 MHz and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ77.00). High resolution mass spectrometry (HRMS) was performed using MALDI in either α-cyano-4-hydroxycinnamic acid or 3,5-dimethoxy-4-hydroxycinnamic acid matrices. Low resolution mass spectrometry (LRMS) was performed using ESI.Ib. Aziridine and Aziridinyl Peptide Synthesisi. Synthesis of Ac-Azy(Me)-NHBn (7)
H-Azy(Me)-NHBn (6) Tr-Azy(Me)-NHBn1a (S1, 860 mg, 1.99 mmol) was added to an ice-cold stirring solution of 1:1 CHCl3/MeOH (7 mL) and stirred until homogeneous. TFA (3.0 mL, 39 mmol) was added dropwise to the stirring solution over 10 minutes. After 2 h, the reaction was diluted with EtOAc (250 mL) and extracted with water (3×100 mL). The combined aqueous extracts were neutralized by adding portions of sat. NaHCO3 until solution reached pH 8 (litmus). The aqueous solution was extracted with DCM (3×100 mL) and the DCM layers were combined, dried (MgSO4), filtered, and solvent was removed under reduced pressure to afford 6 as clear oil that solidified when stored at 4° C. (360 mg, 95% yield). This solid was used without further purification. Rf 0.24 (6% MeOH/DCM); HPLC: gradient 5% to 70% MeCN/H2O over 20 min, 0.5 mL/min tR: 12.8 min; 1H NMR (300 MHz, CDCl3) δ 7.33-7.23 (5H), 6.92 (bs, 1H), 4.42 (d, J=6.0 Hz, 2H), 2.70 (d, J=6.7 Hz, 1H), 2.38 (m, 1H), 1.29 (bs, 1H), 1.13 (d, J=5.7 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 169.4, 138.4, 128.8, 128.0, 127.6, 43.3, 36.6, 32.5, 13.8; HRMS m/z calcd for C11H15N2O [MH+] 191.1263. found 191.1196.
Ac-Azy(Me)-NHBn (7). A genuine sample of 7 was prepared as follows: To a stirring solution of 6 (46 mg, 0.24 mmol) in DCM (1 mL) was added DIEA (0.200 mL, 1.15 mmol) and Ac2O (0.050 mL, 0.53 mmol). After stirring at rt for 1 h, the reaction mixture was directly loaded onto a silica gel column for chromatographic purification (6% MeOH/DCM) to afford the desired product as a clear, colorless oil (52 mg, 93% yield). Rf 0.28 (6% MeOH/DCM); HPLC: gradient 5% to 70% MeCN/H2O over 20 min, 0.5 mL/min tR: 14.3 min; 1H NMR (300 MHz, CDCl3) δ 7.40-7.14 (5H), 6.65 (s, 1H), 4.51 (dd, J=14.7, 6.3 Hz, 1H), 4.36 (dd, J=14.6, 5.6 Hz, 1H), 3.16 (d, J=6.8 Hz, 1H), 2.80-2.70 (m, 1H), 2.10 (s, 3H), 1.25 (d, J=5.7 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 182.2, 166.5, 137.9, 129.0, 128.1, 127.9, 43.5, 41.9, 38.6, 23.6, 13.6; HRMS m/z calcd for C13H17N2O2 [MH+] 233.1290. found 233.1102.ii. Synthesis of Ac-Phe-Azy(Me)-NHBn (9)
Ac-Phe-Azy(Me)-NHBn (9). A genuine sample of 9 was prepared as follows: Ac-Phe-OH (S2, 50 mg, 0.24 mmol)+HOBt (40 mg, 0.30 mmol)+6 (44 mg, 0.23 mmol) were stirred into a suspension in DCM (0.5 mL) at rt. To make the reaction homogenous, a small amount of DMF (˜0.3 mL) was added. To the stirring solution was added DIC (50 μL, 0.32 mmol). After 1 h, the resulting white suspension was filtered and the filter cake was washed with EtOAc (10 mL). The filtrate was washed with water (6×10 mL), and the organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. Flash chromatographic purification (6% MeOH/DCM) of the crude residue afforded 9 as a white solid (30 mg, 34% yield). Rf 0.18 (6% MeOH/DCM); HPLC: gradient 15% to 50% B in A over 25 min, 0.5 mL/min tR: 22.0 min; 1H NMR (300 MHz, CDCl3) δ 7.41-7.01 (10H), 6.34 (bm, J=5.8, 1H), 6.23 (bm, 1H), 4.59 (ddd, J=9.6, 7.1, 5.9, 1H), 4.46 (dd, J=14.7, 6.6, 1H), 4.27 (dd, J=14.7, 5.5, 1H), 3.09 (dd, J=13.0, 5.8, 1H), 2.98 (dd, J=13.0, 9.6, 1H), 2.89-2.79 (m, 2H), 1.97 (s, 3H), 1.13 (d, J=6.5, 3H); 13C NMR (75 MHz, CDCl3) δ 183.1, 170, 4, 166.3, 138.1, 136.3, 129.5, 128.9, 128.8, 128.1, 127.8, 127.6, 56.6, 43.4, 41.1, 39.1, 38.7, 23.0, 13.4. HRMS m/z calcd for C22H26N3O3 [MH+] 380.1974. found 380.1980.iii. Synthesis of H-Azy(Me)-Phe-Gly-NH2 (20)
Fmoc-Phe-Gly-NH2 (S5). To a stirring solution of Fmoc-Phe-OH(S3, 1.800 g, 4.647 mmol)+HATU (1.856 g, 4.881 mmol) in DMF (19.0 mL) was added DIEA (2.40 mL, 13.8 mmol). This solution slowly changed from colorless to yellow over 5 minutes, when the HCl salt of H-Gly-NH2 (S4, 0.444 g, 4.02 mmol) was added and the reaction stirred under dry argon for 3 h. The reaction was subsequently concentrated under high vacuum to ˜5 mL, then diluted with EtOAc (500 mL). The resulting yellow solution was sequentially washed with 10% aq. citric acid, sat. NaHCO3, and brine (100 mL each). The organic layer was dried (MgSO4), filtered, and left to stand at rt whereupon S5 began to spontaneously crystallize. After allowing the mixture to stand overnight, a first crop was collected and air-dried (0.949 g, 53% yield, mp 185-187° C.). After successive concentrations and recrystallizations of the mother liquor, subsequent crops had elevated, broader melting ranges (528 mg combined, 28%, mp 195-201° C.). These samples were identical to the first crop by 1H NMR analysis. Rf 0.18 (7% MeOH/DCM); 1H NMR (300 MHz, DMSO-d6) δ 8.22 (t, J=5.6, 1H), 7.86 (d, J=7.5, 2H), 7.72-7.57 (overlapped d+t, 3H), 7.45-7.06 (13H), 4.24 (ddd, J=10.4, 8.6, 4.1, 1H), 4.19-4.04 (3H), 3.68 (dd, J=17.0, 5.9, 1H), 3.61 (dd, J=17.0, 5.9, 1H), 3.03 (dd, J=13.6, 4.1, 1H), 2.77 (dd, J=13.6, 10.6, 1H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 171.4, 156.6, 144.4, 141.3, 138.9, 129.9, 128.7, 128.3, 127.7, 126.9, 126.0, 120.8, 66.4, 56.9, 47.2; HRMS m/z calcd for C26H26N3O4 [MH+] 444.1923. found 444.1722; m/z calcd for C26H25N3NaO4 [MNa+] 466.1743. found 466.1622.
Tr-Azy(Me)-Phe-Gly-NH2 (S9). S6 (140 mg, 0.316) was subjected to a standard Fmoc deprotection procedure (see section Ic-ii below) and then and then coupled to Tr-Azy(Me)-OH1b (S8) using the standard HATU coupling procedure (see section Ic-ii). The reaction was diluted with EtOAc (20 mL) and washed sequentially with 10% citric acid (15 mL), water (6×15 mL), and brine (20 mL). The organic layer was dried (MgSO4), filtered, and solvent was removed under reduced pressure. The crude product was purified by flash chromatography (7% MeOH/DCM) to afford S9 as a white foam (83 mg, 48% over two steps). Rf 0.57 (10% MeOH/DCM); FIRMS m/z calcd for C34H35N4O3 [MH+] 547.2709. found 547.2928.
H-Azy(Me)-Phe-Gly-NH2 (24).Method A: To an ice-cold stirring solution of 1:1 CHCl3/MeOH (1 mL)+S9(29 mg, 0.053 mmol) was added TFA (3.0 mL, 39 mmol, dropwise over 5 minutes). After 30 minutes, solvent was removed by rotary evaporation (ca. 20 Torr, 22° C.) to afford an oily yellow solid. Et2O (1 mL) was added to the flask to precipitate a slightly sticky white solid. The supernatant was decanted carefully by Pasteur pipette, and the Et2O wash/decant was repeated twice. The white solid had residual solvent removed in vacuo to afford 24 as it is TFA salt (18 mg, 81%). This salt, showing >90% purity by HPLC, was used directly in the subsequent ligation. Analytical samples of the TFA salt of 24 prepared in H2O or MeOH had to be analyzed immediately after being prepared or significant decomposition would be observed.Method B: The reaction was performed as in method A. The oily yellow solid obtained from rotary evaporation is partitioned between EtOAc (1 mL) and sat. NaHCO3 (3×1 mL). The combined aq. layers were washed with Et2O (2 mL). Residual volatile organics were removed by a brief rotary evaporation. The crude product solution was purified by preparative HPLC to afford 24 as a fluffy white solid (10.1 mg, 61% yield). This lyophilized product was found to be stable to storage in the freezer for 5 days, however when analyzed again after 2 months significant decomposition was observed.
HPLC: gradient 5% to 95% B in A over 25 min, 0.8 mL/min tR: 6.9 min; 1H NMR (600 MHz, DMSO-d6) δ 8.71 (d, J=7.9 Hz), 8.30 (t, J=5.7 Hz, 1H), 8.25 (t, J=5.8 Hz), 7.35-7.03 (8H), 4.54 (m, 1H), 3.68 (m, 1H), 3.58 (m, 1H), 3.05 (dd, J=14.0, 4.5 Hz, 1H), 2.84 (dd, J=13.9, 9.9 Hz, 1H), 2.37 (dd, J=8.6, 6.8 Hz, 1H), 2.19-2.03 (m, 1H), 1.08 (d, J=5.7 Hz, 1H), 0.53 (d, J=5.5 Hz, 2H); HRMS m/z calcd for C15H21N4O3 [MH+] 305.1614. found 305.1669; m/z calcd for C15H20N4NaO3 [MNa+] 327.1433. found 327.1539.
Ic. Thioacid Synthesis
i. Synthesis of Ac-Phe-SH (8)
Ac-Phe-OSu (S10). An ice-cold stirring suspension of Ac-Phe-OH(S2, 0.504 g, 2.43 mmol) in DCM (10 mL) was sequentially charged with DCC (0.187 g, 2.95 mmol) and N-Hydroxysuccinimide (NHS, 307 mg, 2.67 mmol). The reaction was stirred for 3.5 h at 0° C., when 1H NMR analysis of the crude reaction mixture showed the reaction to be complete (diagnostic peak for remaining S2 (300 MHz, CDCl3) δ 1.82 ppm). The resulting white sus-pension was filtered and the filter cake washed with DCM (10 mL). The filtrate was chilled in a −10° C. freezer for 2 h and filtered again with minimal DCM rinsing of the filtercake (1-2 mL). The DCM solution was concentrated by rotary evaporation and the product was precipitated from EtOAc to afford S10 as a white solid (734 mg, 99% yield). This product, contaminated with a neglible amount of dicyclohexylurea, was used without further purification. 1H NMR (300 MHz, CDCl3) δ 7.42-7.10 (5H), 5.78 (d, J=7.7 Hz, 1H), 5.29 (m, 1H), 3.33 (dd, J=14.1, 6.1 Hz, 1H), 3.23 (dd, J=14.2, 5.6 Hz, 1H), 2.86 (s, 4H), 1.97 (s, 3H).; HRMS m/z calcd for C15H16N2NaO5 [MNa+] 327.0957. found 327.3312.
Ac-Phe-SH (8). Following the general procedure reported by Gelb,2 to a stirring solution of S10 (113 mg, 0.371 mmol) in MeOH (3.0 mL) was added with NaSH hydrate (pellet form, 59 mg, 1.0 mmol) at rt. The NaSH dissolved within 5 minutes, resulting in a translucent yellow solution that gradually changed to a yellow opaque suspension during the course of the reaction. After stirring for 2.5 h, the reaction has solvent removed by rotary evaporation and the resulting residue was partitioned between water (5 mL) and EtOAc (2×5 mL). The aq. layer was acidified by dropwise addition of conc. HCl solution (−0.2 mL) to pH 1 (litmus). The acidic layer was extracted with DCM (3×10 mL), and the combined organic extracts were dried (MgSO4), filtered, and solvent was removed under reduced pressure to afford 8 as a colorless oil (52 mg, 63% yield). The product was found to be very unstable to a variety of storage conditions, thus was used immediately in the subsequent ligation reactions. 1H NMR (300 MHz, CDCl3) δ 7.46-7.07 (5H), 6.24 (bs, 1H), 4.92 (dt, J=7.5, 5.9, 1H), 3.17 (dd, J=14.2, 5.7, 1H), 3.04 (dd, J=14.3, 7.3, 1H), 1.96 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 199.1, 170.6, 135.6, 129.5, 129.0, 128.8, 127.6, 61.1, 37.6, 25.7, 23.2.ii. General Fmoc-Based Solution Phase Peptide Synthesis Protocols.
General Fmoc deprotection procedure of amino acid derivatives: The Fmoc-protected compound was dissolved and stirred in a solution of DCM (0.1 M)+DBU (2 equiv.) at rt. The reaction, unless otherwise noted, was complete within 30 min. The reaction mixture was directly loaded onto a short silica gel column (50 mL dry silica/1 g Fmoc-protected starting material) with minimal DCM rinsing. The column was eluted under pressure (as in flash chromatography, but with the solvent flow rate increased ˜300%) with DCM to remove dibenzofulvene and subsequently eluting with 5-10% MeOH in DCM to obtain the free amine. The product had solvent removed under reduced pressure and was used directly in the subsequent coupling step.
General HATU peptide coupling procedure: Reactions were performed using a modified version of Carpino's original procedure:3 An ice cold stirring solution of the carboxylic acid (1.2 equiv.) in DMF (0.1 M) was sequentially charged with HATU (1.2 equiv.) and DIEA (1.2 equiv.). The resulting solution was stirred for 5 minutes. The free amine (1.0 equiv.) was added as a solution DMF. The ice bath removed after 0.5 h, and the reaction was monitored by TLC for the disappearance of the free amine. Once complete, the reaction was worked up and the product purified as indicated.
iii. Synthesis of Fmoc-Phe-Ala-SH (15).
Fmoc-Phe-Ala-OMe (S13). Fmoc-Phe-OH(S11, 831 mg, 2.15 mmol)+HOBt (295 mg, 2.18 mmol) was stirred into an ice-cold suspension in DCM (20 mL). The reaction was charged with DIC (0.340 mL, 2.20 mmol) and stirred for 5 minutes. To the resulting homogenous colorless solution was added the HCl salt of H-Ala-OMe (S12, 251 mg, 1.80 mmol) and DIEA (0.320 mL, 1.84 mmol). After 70 minutes, the resulting suspension was filtered, the filtercake was washed with DCM (10 mL), and the combined filtrates were chilled to −10° C. for 1 h. The mixture was filtered again, then washed with sat. NaHCO3 (2×10 mL) and brine (10 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was precipitated from Et2O and residual solvent was removed under reduced pressure, affording S13 as a white solid (983 mg, 97% yield). The crude product was used without further purification despite a minor contamination of diisopropylurea. An analytical sample was obtained by flash chromatographic purification (35% EtOAc/hexanes). Rf 0.59 (1:1 EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J=7.5 Hz, 2H), 7.54 (m, 2H), 7.40 (t, J=7.2 Hz, 2H), 7.35-7.15 (7H), 6.29 (d, J=5.6 Hz, 1H), 5.35 (d, J=6.3 Hz, 1H), 4.55-4.41 (overlapped m, 3H), 4.33 (t, J=8.7 Hz, 1H), 4.19 (t, J=6.9 Hz, 1H), 3.71 (s, 3H), 3.14 (dd, J=13.5, 6.2 Hz, 1H), 3.03 (dd, J=13.1, 7.1 Hz, 1H), 1.34 (d, J=7.2 Hz, 1H); HRMS m/z calcd for C28H27N2O5 [MH+] 473.2067. found 473.1988; m/z calcd for C28H26N2NaO5 [MH+] 495.1896. found 495.1872.
Fmoc-Phe-Ala-OSu (S14)+Fmoc-Phe-ala-OSu (epi-S14). To a stirring solution of S13 (483 mg, 1.02 mmol) in 4:1 Me2CO/H2O at rt was added 2M aq. NaOH (1.0 mL, 2.0 mmol). After 1 h, TLC analysis indicated the consumption of S13 along with the formation of dibenzolfulvene, indicating partial decomposition of the Fmoc group during this process. Acetone was removed by rotary evaporation, and the aq. layer was washed with Et2O (5 mL). The aq. layer was carefully acidified to pH 1 with conc. HCl (−0.5 mL), then extracted with DCM (2×20 mL). The DCM extracts were combined, dried (MgSO4), filtered, and had solvent removed under reduced pressure to afford the crude carboxylic acid (231 mg, 49% yield).4 The residue was stirred into solution in DCM (10 mL). To the stirring solution was added DCC (170 mg, 0.824 mmol)+NHS (89 mg, 0.77 mmol). With intent to racemize the product at this stage, the reaction was left to stir overnight at rt. The resultant white suspension was filtered, and the filtercake was washed with DCM (10 mL). The combined filtrates were chilled to −10° C. for 1 h. The mixture was filtered again, and solvent was removed by rotary evaporation. Stirring the resulting sticky foam with Et2O/hexanes (1:1, ˜4 mL) precipitated a white solid that was significantly easier to handle. Residual solvent was removed under reduced pressure to afford S14 and epi-S14 (285 mg, 49% yield over two steps). As the product is an inseparable mixture, only diagnostic peaks of the 1H NMR are being reported. 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J=7.5 Hz, 1H), 7.53 (dd, J=7.1, 4.3 Hz, 1H), 7.40 (t, J=7.1 Hz, 1H), 2.86 (s), 2.83 (s), 2.82 (s).
Fmoc-Phe-Ala-SH (15)+Fmoc-Phe-Ala-SH (epi-15). The mixture of S14+epi-S14 (102 mg, 0.184 mmol) was dissolved in MeOH (2 mL). To the stirring solution was added NaSH hydrate (29 mg, 0.52 mmol). The reaction changed from a colorless solution to a semi-transparent yellow suspension over the course of 2.5 h. At this time, solvent was removed by rotary evaporation and the residue was partitioned between H2O (10 mL) and Et2O (10 mL). After removing trace volatiles by rotary evaporation, a white precipitate formed in the aq. layer. This precipitate was brought back into solution by addition of a small amount of MeOH. The crude product solution was purified by preparative HPLC (in this case, lyophilization required the use of tBuOH as a co-solvent) to afford 15 (18.4 mg, 21% yield) and epi-15 (3.0 mg, 3% yield). Significant quantities of side-products were observed but were not identified.4 
15: HPLC: gradient 5% to 100% B in A for 20 min, 0.6 mL/min; tR: 18.0 min; 1H NMR (600 MHz, DMSO-d6) δ 8.54 (bs, 1H), 7.87 (d, J=7.5 Hz, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.67-7.60 (m, 2H), 7.42-7.25 (8H), 7.18 (t, J=7.4 Hz, 1H), 4.37-4.30 (m, 1H), 4.27 (ddd, J=11.9, 8.9, 3.4 Hz, 1H), 4.12 (s, 3H), 3.11 (dd, J=13.8, 3.2 Hz, 1H), 2.76 (dd, J=13.8, 11.4 Hz, 1H), 1.30 (d, J=7.1 Hz, 3H); HRMS m/z calcd for C27H26N2NaO4S [MNa+] 497.1511. found 497.0669.
epi-15: HPLC: gradient 5% to 100% B in A for 20 min, 0.6 mL/min; tR: 17.6 min; HRMS m/z C27H26N2NaO4S [MNa+] 497.1511. found 497.1478.
iv. Synthesis of H-Lys-Tyr-Thr-SH (17).
Fmoc-Tyr(tBu)-Thr(tBu)-STmb (S16). S155 (469 mg, 0.790 mmol) was subjected to the standard Fmoc deprotection procedure and then coupled to Fmoc-Tyr(tBu)-OH using the standard HATU coupling procedure. The reaction was diluted with EtOAc (150 mL), and washed with 10% citric acid solution (50 mL), sat. NaHCO3 (3×50 mL), and brine (50 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified by flash chromatography (dry loading the crude product onto silica is strongly recommended; eluted with 40% EtOAc/hexanes) to afford S16 as a white foam (437 mg, 68% yield over two steps).6 Rf 0.62 (1:1 EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.81-6.80 (14H), 6.05 (s, 1H), 5.32 (d, J=8.9, 1H), 4.59-4.41 (m, 2H), 4.27 (m, 2H), 4.16 (m, 2H), 3.75 (bs, 9H), 3.17 (dd, J=13.7, 5.1, 1H), 3.05 (dd, J=14.1, 6.9, 1H), 1.29 (s, 9H), 1.10 (s, 9H); HRMS m/z calcd for C46H56N2NaO9S [MNa+] 835.3604. found 835.2576.
Fmoc-Lys(Boc)-Tyr(tBu)-Thr(tBu)-STmb (S17). S16 (420 mg, 0.517 mmol) was subjected to the standard Fmoc deprotection procedure and then coupled to Fmoc-Lys(Boc)-OH using the standard HATU coupling procedure. The reaction was diluted with EtOAc (150 mL), and washed with 10% citric acid solution (50 mL), sat. NaHCO3 (3×50 mL), and brine (50 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified using flash chromatography (dry loading the crude product onto silica is strongly recommended, eluted with 1:1 EtOAc/hexanes) to afford S17 as a white foam (413 mg, 77% yield over two steps). Rf 0.29 (1:1 EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.81-6.41 (m, 15H), 6.08 (s, 2H), 4.86-4.64 (m, 2H), 4.47 (d, J=8.9, 3H), 4.43-4.14 (m, 6H), 3.79 (s, 3H), 3.77 (s, 6H), 3.20 (dd, J=14.4, 5.9, 1H), 3.02 (m, 3H), 1.83-1.67 (m, 1H), 1.59 (m, 1H), 1.42 (s, 9H), 1.27 (s, 9H), 1.10 (s, 9H); HRMS m/z calcd for C57H76N4NaO12S [MNa+] 1063.5078. found 1063.4727.
H-Lys-Tyr-Thr-SH (17). S17 (48 mg, 0.046 mmol) was subjected to the standard Fmoc de-protection procedure. The amine residue was chilled to 0° C., and the reaction vessel was purged under high vacuum and flushed with argon. An ice-cold solution of TFA (0.600 mL), DCM (0.200 mL), and Et3SiH (0.250 mL) was charged to the reaction vessel and stirred at 0° C. After 0.5 h, the ice bath was removed and the reaction allowed to warm to ambient temperature, where it was monitored by HPLC and LRMS until analysis showed the reaction to be complete after an additional 3.5 h. Diagnostic HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 5.5 min for 17, tR: 17.4 min for H-Lys-Tyr(OH)-Thr(OH)-STmb (identified by LRMS: m/z calcd for C29H43N4O8S [MH+] 607.1. found 607.1). The reaction was partitioned between water (10 mL) and Et2O (2×6 mL). The aqueous layer had trace volatiles removed by brief rotary evaporation, and then the crude product solution was purified by preparative HPLC to afford the bis-TFA salt of 17 as a fluffy white solid (22 mg, 73% yield over two steps). HRMS m/z calcd for C19H30N4O5S [MH+] 427.2015. found 427.2891.v. Synthesis of H-Glu-Tyr-Thr-SH (19)
Fmoc-Glu(tBu)-Tyr(tBu)-Thr(tBu)-STmb (S18). S16 (285 mg, 0.315 mmol) was subjected to the standard Fmoc deprotection procedure and then coupled to Fmoc-Glu(tBu)-OH using the standard HATU coupling procedure. The reaction was diluted with EtOAc (100 mL), and washed with 10% citric acid solution (30 mL), sat. NaHCO3 (2×30 mL), water (20 mL) and brine (30 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified using flash chromatography (dry loading the crude product onto silica is strongly recommended, eluted with 45% EtOAc/hexanes) to afford S18 as a white foam that reverted to an clear oil upon standing at rt. Triturating the oil at −78° C. with Et2O/hexanes (˜1:10 mixture) and removal of solvent under reduced pressure afforded S18 as a white powder (340 mg, 97% yield over two steps). Rf 0.50 (1:1 EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J=7.3, 2H), 7.59 (d, J=7.6, 2H), 7.39 (t, J=7.4, 2H), 7.31 (t, J=7.5, 2H), 7.13 (d, J=8.4, 2H), 6.96 (d, J=7.2, 1H), 6.84 (d, J=8.4, 2H), 6.71 (d, J=9.2, 1H), 6.08 (s, 2H), 5.64 (d, J=7.5, 1H), 4.71 (˜q, J=7.0, 1H), 4.47 (d, J=8.9, 1H), 4.35 (d, J=6.9, 2H), 4.27 (d, J=7.2, 1H), 4.21 (d, J=6.3, 1H), 4.16 (d, J=2.9, 1H), 3.79 (s, 3H), 3.77 (s, 6H), 3.21 (dd, J=14.2, 5.6, 1H), 3.01 (dd, J=14.1, 7.4, 1H), 2.47-2.20 (2H), 2.11-1.95 (1H), 1.87 (2H), 1.44 (s, 9H), 1.27 (s, 9H), 1.10 (s, 9H).; HRMS m/z calcd for C55H71N3NaO12S [MNa+] 1020.4656. found 1020.4389.
H-Glu-Tyr-Thr-SH (19). S18 (63.5 mg, 0.0636 mmol) was subjected to the standard Fmoc de-protection procedure. The amine residue was chilled to 0° C., and the reaction vessel was purged under high vacuum and flushed with argon. An ice-cold solution of TFA (0.600 mL), DCM (0.200 mL), and Et3SiH (0.250 mL) was charged to the reaction vessel and stirred at 0° C. After 0.5 h, the ice bath was removed and the reaction was allowed to warm to ambient temperature, where it was monitored by HPLC and LRMS until analysis showed the reaction to be complete after an additional 3.5 h. The reaction was partitioned between water (13 mL) and Et2O (2×6 mL). The aqueous layer had trace volatiles removed by brief rotary evaporation, and then the crude product solution was purified by preparative HPLC to afford the TFA salt of 19 as a fluffy white solid (18.2 mg, 53% yield over two steps). HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 7.6 min; HRMS m/z calcd for C18H26N3O7S [MH+] 428.1491. found 428.1364.vi. Synthesis of H-Cys-Tyr-Ala-SH (21).
Boc-Ala-STmb (S21). To an ice-cold stirring suspension of S19 (377 mg, 1.99 mmol)+S207 (427 mg, 1.99 mmol)+HOBt (323 mg, 2.39 mmol) in dry DCM (5 mL, fresh dist. under argon from CaH2) under argon was added DIC (302 mg, 2.39 mmol). The white suspension rapidly changed to a homogenous solution that was allowed to stir overnight and warm to ambient temperature. After 24 h (TLC analysis still showed unreacted S20), the reaction was diluted with DCM (5 mL) and washed with NaHCO3 (2×10 mL), water (2×10 mL), and brine. The organic layer was dried (Na2SO4), filtered, and was concentrated by rotary evaporation. The crude residue was purified by flash chromatography (1:4 EtOAc/hexanes) afforded the desired product S21 (167 mg, 22%) as well as unreacted S20.4 Rf 0.67 (30% EtOAc/hexanes); NMR (300 MHz, CDCl3) δ 6.11 (s, 2H), 5.03 (d, J=7.9, 1H), 4.43 (p, J=7.0, 1H), 4.21 (s, 2H), 3.82 (s, 3H), 3.81 (s, 6H), 1.45 (s, 9H), 1.40 (d, J=7.0, 3H); 13C NMR (75 MHz, CDCl3) δ 161.1, 159.4, 104.7, 90.7, 56.4, 56.0, 55.6, 28.6, 22.4, 19.6; HRMS m/z calcd for C18H27NNaO6S [MNa+] 408.1457. found 408.1393.
Fmoc-Tyr(tBu)-Ala-STmb (S22). To a stirring solution of S21 (365 mg, 0.950 mmol) in DCM (6 mL) was added TFA (4 mL). After stirring for 10 minutes, the reaction was washed with a solution of water (10 mL)+20% aq. NaHCO3 (20 mL), then washed once more with 20% aq. NaHCO3 (20 mL). The organic layer was dried (MgSO4), filtered, and had solvent removed under reduced pressure. The free amine residue was used directly in a standard HATU coupling with Fmoc-Tyr(tBu)-OH. The coupling reaction was worked up by diluting with DCM (15 mL) and washing sequentially with sat. NaHCO3 (2×15 mL), water (3×20 mL), and brine (20 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. Flash chromatographic purification of the crude residue afforded S22 as a white solid (509 mg, 74% yield). Rf 0.82 (5% MeOH/DCM); 1H NMR (300 MHz, CDCl3) δ 7.62-6.83 (12H), 6.52 (d, J=6.7, 1H), 6.08 (s, 2H), 5.43 (d, J=8.2, 1H), 4.68 (p, J=7.1 Hz, 1H), 4.41 (m 2H), 4.30 (dd, J=10.6, 6.9, 1H), 4.24-4.11 (m, 3H), 3.77 (s, 9H), 3.05 (d, J=5.6, 2H), 1.34 (d, J=7.0, 3H), 1.30 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 200.7, 170.6, 161.1, 159.4, 154.6, 143.9, 141.5, 130.1, 127.9, 127.3, 125.3, 124.6, 120.2, 90.6, 78.6, 67.3, 56.0, 55.5, 55.1, 47.3, 38.0, 29.0, 22.5, 19.4; FIRMS m/z calcd for C41H46N2NaO8S [MNa+] 749.2873. found 749.2454.
Boc-Cys(Tr)-Tyr(tBu)-Ala-STmb (S23). S22 (249 mg, 0.34 mmol) was subjected to the standard Fmoc deprotection procedure and then coupled to Boc-Cys(Tr)-OH using the standard HATU coupling procedure. The reaction was diluted with DCM (10 mL), and sequentially washed with sat. NaHCO3 (2×10 mL), water (3×15 mL), and brine (10 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude residue was purified by flash chromatography (1:1 EtOAc/hexanes) to afford S27 as a white solid (167 mg, 51%). HS-Tmb (S20, 28 mg, 38%) was recovered as a by-product, leading us to conclude that significant decomposition of the thioester occurred during the Fmoc deprotection procedure.6 Rf 0.28 (30% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.52-7.15 (15H), 7.05 (d, J=8.5, 1H), 6.83 (d, J=8.5, 1H), 6.63 (d, J=6.4, 2H), 6.48 (d, J=8.1, 2H), 6.09 (s, 2H), 4.69 (d, J=6.7, 1H), 4.58 (p, J=7.3, 2H), 4.19 (d, J=3.7, 2H), 3.79 (s, 3H), 3.77 (s, 6H), 3.02 (dd, J=13.3, 6.1, 1H), 2.55 (dd, J=13.0, 5.2, 1H), 1.38 (s, 9H), 1.31 (s, 9H), 1.25 (d, J=1.8, 3H). 13C NMR (75 MHz, CDCl3) δ 200.6, 170.3, 170.0, 161.0, 159.3, 154.5, 144.4, 131.1, 130.0, 129.6, 128.2, 127.1, 124.3, 104.5, 90.6, 80.5, 78.4, 67.4, 55.9, 55.4, 55.1, 54.1, 36.8, 33.7, 29.0, 28.3, 22.3, 18.9; HRMS m/z calcd for C53H63N3NaO9S2 [MNa+] 972.3903. found 972.3837.
H-Cys-Tyr-Ala-SH (21). Solid S23 (39 mg, 0.041 mmol) placed under argon and chilled to 0° C. in an ice-bath. The solid was dissolved in an ice cold solution of TFA (0.600 mL)+DCM (0.150 mL)+Et3SiH (0.250 mL) and stirred into solution. After 6 h, the reaction had reached rt, and HPLC analysis showed the deprotection to be complete. Diagnostic HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 9.9 min for 21, tR: 20.5 min for H-Cys-Tyr-Ala-STmb (identified by LRMS analysis: m/z calcd for C25H33N3NaO7S2 [MNa+] 574.2. found 574.2). It was noted that the HPLC peak belonging to 21 had a small shoulder, and despite our best efforts, better resolution could not be obtained by HPLC. MS analysis of the peak showed no MS peaks other than that assigned to 21. This may be an analytical artifact emanating from its polyfunctional nature or indicate that 21 was partially epimerized during the final global deprotection step (1H NMR of all precursors to 21 show no evidence of an epimer). The reaction was partitioned between water (10 mL) and Et2O (2×6 mL). The aqueous layer had trace volatiles removed by brief rotary evaporation, and then the crude product solution was purified by preparative HPLC to afford the TFA salt of 21 as a fluffy white solid (9 mg, 45% yield). HRMS m/z calcd for C15H22N3O4S2 [MH+] 379.1052. found 379.0930; m/z calcd for C15H21N3NaO4S2 [MNa+] 394.0871. found 394.0649.Id. Aziridine+Thioacid Ligations
Preparation of aqueous buffers. Stock solutions of 2.0 M Na2HPO4 in water and 1.0 M citric acid in water were combined in proportions to give phosphate-citrate buffer of the desired pH.8 The urea-phosphate buffer was prepared by dissolving urea and Na2HPO4 in water, adjusting the pH by adding solid NaOH before bringing the solution up to the desired volume. Using HCl to adjust the pH can lead to undesired decomposition of the aziridine intermediate, thus should be avoided.
i. Reactions to Define Coupling Conditions.
General Procedure for the Metal Mediated Couplings of Thioacetic acid to 6 (Table 3). To a solution of 6 (0.100 mmol) in a 1:1 mixture of DMF and aq. buffer (0.400 mL) was added the appropriate metal salt (1.0 equiv.) and was stirred until homogeneous (in the case of CuCN and CuI, the solution never became homogeneous). To this stirring solution was added AcSH (5, 1.2 equiv.). After 30 minutes, the reaction was filtered through a cotton/celite plug with EtOAc (5 mL) and washed with water (3×5 mL). The organic phase was dried (MgSO4), filtered, and the solvent was removed under reduced pressure. The crude product (85-97% isolated yield) was analyzed by HPLC without further purification. Replicate experiments of entries 1-4, 6, and 10 were performed under argon in degassed solvents. These experiments yielded no significantly different data than obtained from the experiments conducted under the ambient atmosphere.
TABLE 3Initial Experiments to Define the Coupling ReactionRing-EntryMetal saltSolvent6[a]7[a]Opening[a]1Cu(OAc)2•H2O1:1 DMF-buffer0%100%0%(100 mol %)(pH 7.2)2Cu(OAc)2•H2O1:1 DMF-buffer0%100%0%(100 mol %)(pH 6.2)3Cu(OAc)2•H2O1:1 DMF-buffer0%100%0%(100 mol %)(pH 5.2)4Cu(OAc)2•H2O1:1 DMF-buffer0%100%0%(100 mol %)(pH 4.2)5None1:1 DMF-buffer6%13%81%(pH 7.2)6AgOAc1:1 DMF-buffer65%35%0%(100 mol %)(pH 7.2)7K3Fe(CN)61:1 DMF-buffer0%50%50%(100 mol %)(pH 7.2)8CuI1:1 DMF-buffer45%26%29%(pH 7.2)9CuCN1:1 DMF-buffer55%28%17%(pH 7.2)10Cu(OAc)2•H2O1:1 DMF-buffer26%74%0%(5 mol %)(pH 7.2)aPercentages were determined from the HPLC peak integrations of the crude product mixture. Peak identity was determined by LRMS and/or 1H NMR analysis.ii. General Procedures for Table 3.
General Procedure for Ac-Phe-SH (or Ac-phe-SH)+H-Azy(Me)-NHBn Ligations (Table 3). To a stirring solution of 6 (0.040 mmol) in the indicated solvent system was Cu(OAc)2.H2O (1.0 equiv.) and, if indicated, HOBt (2.0 equiv.). Once the solution became homogeneous, an aliquot of a 0.4 M stock solution of 8 in DMF (1.1 equiv., final reaction concentration 0.1 M) was added, where it was noted the reaction slowly changed color to a dark brown over the next 5 minutes. Thirty minutes after the addition of 8, the reaction was treated with a dropwise addition of aq. NaSH, which precipitated a black solid. Aq. NaSH addition was halted (usually ˜0.3 mL) when it had been determined that no more black solid was forming. The heterogeneous mixture was then filtered through a cotton/Celite plug with EtOAc (5 mL). The organic phase was washed with water (5 mL), dried (MgSO4), filtered, and solvent was removed under reduced pressure to afford a white solid which was analyzed by HPLC and/or 1H NMR without further purification.Identification of epi-9. For identification purposes, mixtures of 8 and ent-8 were prepared by an unoptimized version of the synthesis of 8, starting with Ac-Phe-OH(S2) and Ac-phe-OH (ent-S2), respectively. These mixtures were subjected to an unoptimized version of the general procedure for Ac-Phe-SH+H-Azy(Me)-NHBn ligations. For a comparison of ligations using mixtures enriched in 8 vs. mixtures enriched in ent-8 resulting in mixtures of 9 and epi-9, see 1H NMR data.iii. Procedures for Ligation/Ring-Opening Reactions
Ac-Phe-Thr-NHBn (10). To a stifling solution of 6 (0.040 mmol) in DMF (0.300 mL) was added Cu(OAc)2.H2O (1.0 equiv.) and HOBt (2.0 equiv.). Once the solution became homogeneous and dark green, 8 (0.125 mL, 0.4 M in DMF, 1.2 equiv.) was added. It was noted the reaction slowly changed color over the next 5 minutes. After 30 minutes, the reaction was charged with 10% aq. TFA. After 1.5 h, the reaction was neutralized by adding sat. NaHCO3 and filtered through cotton/Celite with EtOAc (5 mL). The filtrate was washed with sat. NaHCO3 (5 mL), filtered, and solvent was removed by rotary evaporation. The crude product was purified by flash chromatography (10% iPrOH/DCM) to afford 10 (11 mg, 69% yield) as a white solid. Rf 0.14 (10% iPrOH/DCM); 1H NMR (300 MHz, CD3OD) δ 7.40-7.09 (10H), 4.67 (dd, J=9.1, 5.9, 1H), 4.37 (s, 2H), 4.29 (d, J=3.7, 1H), 4.21 (dd, J=6.4, 3.7, 1H), 3.13 (dd, J=17.2, 8.6, 1H), 2.91 (dd, J=13.9, 9.0, 1H), 1.90 (s, 3H), 1.14 (d, J=6.3, 3H); 1H NMR (600 MHz, DMSO-d6) δ 8.18 (d, J=8.2 Hz, 1H), 8.08 (t, J=6.1 Hz, 1H), 7.81 (d, J=8.6 Hz, 1H), 7.33-7.17 (9H), 7.15 (t, J=7.1 Hz, 1H), 4.92 (d, J=4.9 Hz, 1H), 4.59 (ddd, J=10.1, 8.4, 4.4 Hz, 1H), 4.28 (overlapped dd, 2H), 4.16 (dd, J=8.6, 3.7 Hz, 1H), 4.05 (complex, overlapped ddt, 1H), 3.01 (dd, J=14.0, 4.3 Hz, 1H), 2.74 (dd, J=14.0, 10.3 Hz, 1H), 1.73 (s, 3H), 1.01 (d, J=6.4 Hz, 3H). 13C NMR (150.8 MHz, DMSO-d6) δ=172.4, 170.7, 170.0, 162.2, 140.0, 138.8, 129.8, 128.8, 128.7, 127.7, 127.3, 67.0, 59.1, 54.6, 23.1, 20.8; HRMS m/z calcd for C22H28N3O4 [MH+] 398.2080. found 398.2201; m/z calcd for C22H27N3NaO4 [MNa+] 420.1899. found 420.2065.
Fmoc-Phe-Ala-Thr-NHBn (16). To a solution of 6 (4.1 mg, 0.022 mmol)+Cu(OAc)2.H2O (4.6, mg, 0.023 mmol) in DMF (0.177 mL) was added HOBt (0.023 mL, 2.00 M in DMF, 0.042 mmol). To the stirring dark green solution was added 15 (10 mg, 0.021 mmol). The reaction color changed to yellow and eventually a very dark brown over the course of 10 minutes. This aziridine intermediate was not detected by analytical HPLC, but was identified by LRMS analysis of the crude reaction mixture (m/z calcd for C38H38N6NaO6 [MNa+] 653.3. found 653.4). After stirring for 3 h (6 was not detected by LRMS after 1.5 h), the reaction was charged with a solution of TFA (0.060 mL)+H2O (0.500 mL). After 3 h, the reaction was diluted with EtOAc (5 mL) and washed with H2O (3 mL). The organic layer was dried (MgSO4), filtered, and had solvent removed under reduced pressure to afford crude 16 (11.5 mg, 84% yield). HPLC analysis of this crude product determined that the epimerization of this coupling/ring-opening process was 5% (epi-16 was identified by a HPLC co-injection with an authentic sample; see below). The crude product was purified by preparative HPLC (in this case, the HPLC solvent was removed by rotary evaporation, precipitating the product. The product was extracted in DCM and solvent was removed under reduced pressure) to afford 16 as a white solid (9.8 mg, 72% yield) HPLC: gradient 50% to 70% MeCN in H2O over 20 min, 0.6 ml/min; tR: 10.1 min; 1H NMR (600 MHz, DMSO-d6) δ 8.37 (d, J=7.1 Hz, 1H), 8.20 (t, J=5.7 Hz, 1H), 7.93 (d, J=7.3 Hz, 1H), 7.87 (d, J=7.1 Hz, 2H), 7.68-7.57 (m, 3H), 7.48 (t, J=7.5 Hz, 12H), 7.43-7.14 (m, 4H), 4.94 (d, J=5.1 Hz, 1H), 4.43-4.37 (m, 2H), 4.32-4.26 (m 3H), 4.19 (dd, J=8.3, 3.6 Hz, 1H), 4.16-4.08 (m, 3H), 4.05 (dd, J=10.1, 5.3 Hz, 1H), 3.84-3.80 (m, 1H), 3.01 (dd, J=13.8, 2.8 Hz, 1H), 2.76 (dd, J=13.4, 11.4 Hz, 1H), 1.26 (d, J=7.1 Hz, 3H), 1.04 (d, J=6.2 Hz, 3H).; HRMS m/z calcd for C38H40N4NaO6 [MNa+] 671.2846. found 671.3943.
Fmoc-Phe-Ala-Thr-NHBn (epi-16). Following the procedure for preparing Fmoc-Phe-Ala-Thr-NHBn, epi-15 (2.6 mg)+6 (1.1 mg) afforded crude epi-16 (3.7 mg, quant.). An aziridine intermediate identical to that above by LRMS was observed. This crude sample was used to identify the epi-16 found in the crude ligation product of 16 (by analytical HPLC co-injection). HPLC analysis of this crude product determined that the epimerization of this coupling/ring-opening process was 5%. The crude product was purified by preparative HPLC (in this case, the HPLC solvent was removed by rotary evaporation, precipitating the product. The product was extracted in DCM and solvent was removed under reduced pressure) to afford epi-16 as a white solid (2.3 mg, 65% yield). HPLC: gradient 50% to 70% MeCN in H2O over 20 min, 0.6 mL/min; tR: 8.8 min; HRMS m/z calcd for C38H40N4NaO6 [MNa+] 671.2846. found 671.3868.
H-Lys-Tyr-Thr-Thr-NHBn (18) (SEQ ID NO: 1). 6 (1.9 mg, 0.010 mmol)+Cu(OAc)2.H2O (2.3 mg, 0.012 mmol)+HOBt (2.8 mg, 0.021 mmol) were dissolved in DMF (0.200 mL) and stirred at rt until homogeneous and dark green. 17 (bis-TFA salt, 8.0 mg, 0.012 mmol) was added to the reaction mixture, which caused the reaction color to change to yellow and eventually a very dark brown over 10 minutes. HPLC monitoring of the reaction showed complete consumption of 6 in favor of H-Lys-Tyr-Thr-Azy(Me)-NHBn after 2.5 h. Although the aziridine intermediate was not isolated, it was observed by HPLC and LRMS analysis: HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 14.0 min; LRMS m/z calcd for C30H43N6O6 [MH+] 583.2. found 583.3; m/z calcd for C30H42N6NaO6 [MNa+] 605.2. found 605.3. The reaction mixture was charged with a solution of water (0.500 mL)+TFA (0.060 mL). After 4 h, the brown heterogeneous mixture was treated with a dropwise addition of aq. NaSH to precipitate a black solid. The reaction was diluted with H2O, and the solid was removed by filtration through a 0.2 μm syringe filter. The filtrate was purified by preparative HPLC to afford 18 as its bis-TFA salt (6.6 mg, 80% yield). HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 12.3 min; 1H NMR (600 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.54 (d, J=7.7, 1H), 8.30 (d, J=8.2, 1H), 8.25 (t, J=5.8, 1H), 8.02 (bs, 3H), 7.67 (bs, 2H), 7.64 (d, J=8.4, 2H), 7.32-7.16 (5H), 7.09 (d, J=7.9, 2H), 6.63 (d, J=7.9, 2H), 5.17 (d, J=4.2, 1H), 4.98 (d, J=3.8, 1H), 4.65 (bs, 1H), 4.35 (dd, J=8.1, 3.5, 1H), 4.28 (d, J=5.8, 1H), 4.22 (dd, J=8.3, 2.5, 1H), 4.08 (d, J=24.3, 2H), 3.70 (bs, 1H), 2.96 (d, J=11.8, 1H), 2.75-2.64 (m, 3H), 1.68 (dd, J=13.5, 6.4, 1H), 1.48 (p, J=7.5, 1H), 1.35-1.23 (m, 2H), 1.05 (d, J=6.2, 3H), 1.01 (d, J=6.0, 3H); HRMS m/z calcd for C30H45N6O7 [MH+] 601.3350. found 601.3032; m/z calcd for C30H44N6NaO7 [MNa+] 623.3169. found 623.2794.
H-Glu-Tyr-Thr-Thr-NHBn (SEQ ID NO: 2) (20, General Procedure for Table 2, entries 4-6). 6 (1 equiv.)+Cu(OAc)2.H2O (1 equiv.)+HOBt (2 equiv.) were dissolved in the indicated solvent (0.200 mL) and stirred at rt until dark green (using DMF as a solvent resulted in a homogenous solution, however aq. buffers resulted in fine opaque suspensions). 19 (TFA salt, 1 equiv.) was added to the reaction mixture, which caused the reaction color to change to yellow and eventually a very dark brown over 10 minutes. HPLC monitoring of the reaction showed consumption of 6 in favor of H-Glu-Tyr-Thr-Azy(Me)-NHBn within 2 h. Although the aziridine intermediate was not isolated, it was observed by HPLC and LRMS analysis: HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 15.2 min; LRMS m/z calcd for C29H38N5O8 [MH+] 584.2. found 584.0; m/z calcd for C29H37N5NaO8 [MNa+] 606.3. found 606.3). The reaction mixture was directly treated with a solution of water (0.500 mL)+TFA (0.060 mL). When HPLC analysis showed the hydrolysis was complete, the brown heterogeneous mixture was treated with a dropwise addition of aq. NaSH to precipitate a black solid. The reaction was diluted with H2O and the solid was removed by filtration through a 0.2 μm syringe filter. The filtrate was purified by preparative HPLC to afford 20 as its TFA salt (DMF, 69% yield; phosphate-citrate buffer, 71% yield; urea-phosphate buffer, 88% yield). HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 13.3 min; 1H NMR (600 MHz, DMSO-d6) δ 12.27 (s, 1H), 9.18 (s, 1H), 8.53 (d, J=7.9 Hz, 1H), 8.33 (d, J=8.4 Hz, 1H), 8.22 (t, J=6.0 Hz, 2H), 8.02 (d, J=4.3 Hz, 3H), 7.64 (d, J=8.5 Hz, 1H), 7.30-7.15 (5H), 7.10 (d, J=8.5 Hz, 2H), 6.63 (d, J=8.5 Hz, 2H), 5.16 (bs, 1H), 4.95 (bs, 1H), 4.65-4.63 (m, 1H), 4.38 (dd, J=8.4, 4.0 Hz, 1H), 4.28 (d, J=6.2 Hz, 2H), 4.21 (dd, J=8.5, 3.2 Hz, 1H), 4.10 (dd, J=5.7, 3.1 Hz, 1H), 4.06-4.02 (m, 1H), 3.73 (dd, J=10.5, 5.4 Hz, 1H), 2.95 (dd, J=14.0, 3.2 Hz, 1H), 2.65 (dd, J=14.2, 10.6 Hz, 1H), 2.33 (dd, J=8.4, 3.5 Hz, 1H), 2.31 (dd, J=8.1, 3.3 Hz, 1H), 1.92 (dd, J=14.5, 8.0 Hz, 2H), 1.05 (d, J=6.5 Hz, 3H), 1.00 (d, J=6.3 Hz, 3H); HRMS m/z calcd for C29H39N5O9 [MH+] 602.2826. found 602.2706.
Ligation of H-CYA-SH+H-Azy(Me)-NHBn. 6 (2.5 mg, 0.013 mmol)+Cu(OAc)2.H2O (3.6 mg, 0.018 mmol)+HOBt (4.4 mg, 0.033 mmol) were dissolved in DMF (0.200 mL) and stirred at rt until homogeneous and dark green. 21 OVA salt, 7.6 mg, 0.016 mmol) was added to the reaction mixture, which caused the reaction color to change to black immediately. HPLC monitoring of the reaction showed incomplete consumption of 6 in favor of the disulfide dimer (H-Cys-Try-Ala-Azy(Me)-NHBn)2 after 2 h. Although the aziridine intermediate was not isolated, it could be observed by HPLC and LRMS: HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 19.7 min; LRMS m/z calcd for C52H64N10NaO10S2 [MNa+] 1075.4. found 1075.5. It was noted that (1) there was no LRMS evidence for the presence of the H-Cys-Tyr-Ala-Azy(Me)-NHBn and (2) the reaction mixture for this ligation was significantly more complex than the other examples listed in Table 2. The reaction mixture was charged with a solution of water (0.250 mL)+TFA (0.030 mL). After 2.5 h, when the black heterogeneous mixture was treated with a dropwise addition of aq. NaSH to precipitate a black solid. The mixture was diluted with MeOH (7 mL) and the solid was removed by filtration through a 0.2 μm syringe filter. The filtrate was purified by preparative HPLC to afford 22 as its TFA salt (3.5 mg, 40% yield) and 23 as its bis-TFA salt (3.5 mg, 43% yield). Formation of 22 is believed to have been caused by a reduction of the disulfide dimer by the NaSH that was added to precipitate the copper.
22: HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 14.2 min; HRMS m/z calcd for C26H36N5O6S [MH+] 1546.2386. found 546.2922; m/z calcd for C26H36N5NaO6S [MNa+] 568.2206. found 568.2344.
23: HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 17.5 min; 1H NMR (600 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.72 (bs, 1H), 8.51 (d, J=5.7, 1H), 8.29-8.14 (4H), 7.66 (d, J=8.4, 1H), 7.31-7.23 (6H), 7.20 (t, J=7.1, 2H), 7.05 (d, J=8.2, 1H), 6.63 (d, J=8.4, 2H), 4.94 (bs, 1H), 4.57 (bs, 1H), 4.38 (p, J=7.2 Hz, 1H), 4.32 (dd, J=15.4, 6.2, 1H), 4.26 (dd, J=15.3, 5.8, 1H), 4.23 (dd, J=8.5, 3.6, 1H), 4.03 (s, 2H), 2.96-2.91 (2H), 2.66 (dd, J=13.6, 10.2, 1H), 1.22 (d, J=7.1, 3H), 1.02 (d, J=6.4, 3H). LRMS m/z calcd for C52H67N10O12S2 [MH+] 1089.4. found 1089.4; m/z calcd for C26H36N5NaO6S [MNa+] 1089.4. found 1089.4.
Other products: Crude samples of the reaction mixture, both before and after addition of aq. TFA, were analyzed by LRMS, which gave peaks consistent with 21, 22, and 23 (see above). Minor by-products were tentatively identified as follows: observed before addition of aq. TFA: (S24) m/z calcd for C41H50N6NaO9S2 [MNa+] 885.3. found 885.4; (S26) m/z calcd for C41H50N6NaO9S3 [MNa+] 917.3. found 917.4; (S27) m/z calcd for C52H64N10NaO10S3 [MNa+] 1114.3. found 1114.2; Observed after aq. I′M addition: (S25) m/z calcd for C41H52N8O10S2 [MH+] 881.3. found 881.3; (S28) m/z calcd for C52H68N10O12S3 [MH+].
We concluded that these side products emanate from perthioester intermediates. Tam et. al.9 first disclosed the reaction of thiols and thioacids forming perthiolated products, and the LRMS analysis done on this reaction mixture is consistent with a complex mixture of di-sulfide and tri-sulfide products. An 83% combined yield of the desired products 22 and 23 demonstrates that the aziridine mediated ligation is remarkably faster than the observed side reactions that, presumably, the free thiol causes.
H-Lys-Tyr-Thr-Thr-Phe-Gly-NH2 (25) (SEQ ID NO: 4). 24 (5.0 mg, 0.012 mmol)+Cu(OAc)2.H2O (2.5 mg, 0.013 mmol)+HOBt (3.4 mg, 0.025 mmol) were dissolved in DMF (0.200 mL) and was stirred at rt until homogeneous and dark green. To this solution was added 17, which caused the reaction to change to a dark brown over the next 15 minutes. The formation of H-Lys-Tyr-Thr-Azy(Me)-Phe-Gly-NH2 (SEQ ID NO: 6) was observed by HPLC and LRMS analysis: HPLC: isocratic 10% B in A, 2 minutes, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 12.6 min; LRMS m/z calcd for C34H49N8O8 [MH+] 697.4. found 697.5; m/z calcd for C34H50N8NaO9 [MNa+] 719.4. found 719.6. When HPLC analysis showed the coupling to be complete (2 h), the reaction mixture was charged with a solution of TFA (0.060 mL)+H2O (0.500 mL). After 3.5 h, the reaction mixture was treated with aq. NaSH to precipitate a black solid. The solid was removed by flushing the reaction mixture through a 0.2 μm syringe filter with MeOH (10 mL). The filtrate had solvent removed by rotary evaporation and was reconstituted in water for purification by preparative HPLC to afford the TFA salt of 25 as a white solid (8.8 mg, 78% yield). HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A for 23 min, 0.6 mL/min; tR: 11.1 min; 1H NMR (600 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.53 (d, J=8.1, 1H), 8.26 (d, J=8.4, 1H), 8.19 (t, J=5.8, 1H), 8.01 (d, J=7.5, 4H), 7.65 (s, 3H), 7.58 (d, J=8.1, 2H), 7.26-7.03 (m, 9H), 6.63 (d, J=8.5, 2H), 5.07 (d, J=4.5, 1H), 4.97 (d, J=5.0, 1H), 4.69-4.61 (m, 1H), 4.49 (td, J=8.4, 5.3, 1H), 4.32 (dd, J=8.4, 3.8, 1H), 4.21 (dd, J=8.1, 3.9, 1H), 4.03 (qd, J=10.5, 5.5, 2H), 3.70 (bs, 1H), 3.64 (dd, J=16.8, 6.1, 1H), 3.55 (dd, J=16.8, 5.6, 1H), 3.04 (dd, J=14.0, 5.1, 1H), 2.97 (dd, J=14.2, 3.5, 1H), 2.81 (dd, J=13.9, 8.8, 1H), 2.71 (bs, 2H), 2.67 (dd, J=14.5, 10.5, 1H), 1.68 (dd, J=14.4, 7.1, 2H), 1.48 (p, J=7.5, 2H), 1.35-1.22 (m, 2H), 1.00 (d, J=6.3, 3H), 0.99 (d, J=6.3, 3H); HRMS m/z calcd for C34H51N8O9S [MH+] 715.3779. found 715.3059.
H-Glu-Tyr-Thr-Thr-Phe-Gly-NH2 (26) (SEQ ID NO: 5). 24 (5.6 mg, 0.018 mmol)+Cu(OAc)2 (0.019 mmol from an aq. stock solution)+HOBt (0.038 mmol from a 2.00M DMF stock solution) were dissolved in buffer (0.105 mL, 8M Urea, 0.1 M Pi, pH 7.53), and was stirred at rt until homogeneous and dark green. To this solution was added 19 (9.9 mg, 0.018 mmol), which caused the reaction to change to a dark brown immediately. The formation of H-Glu-Tyr-Thr-Azy(Me)-Phe-Gly-NH2 (SEQ ID NO: 7) was observed by HPLC and LRMS analysis: HPLC: isocratic 10% B in A, 2 minutes, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 13.0 min; LRMS m/z calcd for C33H44N7O10 [MH+] 698.1. found 698.2; m/z calcd for C33H43N7NaO10 [MNa+] 720.4. found 720.4. When HPLC analysis showed the coupling to be complete (2 h), the reaction mixture was charged with a solution of TFA (0.060 mL)+H2O (0.500 mL). After 2 h, the reaction mixture was treated with aq. NaSH to precipitate a black solid. The solid was removed by flushing the reaction mixture through a 0.2 μm syringe filter with MeOH (10 mL). The filtrate had solvent removed by rotary evaporation and was reconstituted in water for purification by preparative HPLC to afford the TFA salt of 26 as a white solid (11.4 mg, 77% yield). HPLC: isocratic 10% B in A, 2 min, then gradient 10% to 60% B in A over 23 min, 0.6 mL/min; tR: 11.3 min; 1H NMR (600 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.52 (d, J=7.9 Hz, 1H), 8.29 (d, J=8.5 Hz, 1H), 8.17 (t, J=5.9 Hz, 1H), 8.02-8.00 (m, 4H), 7.61 (d, J=8.1 Hz, 1H), 7.27-7.03 (m, 9H), 6.63 (d, J=8.5 Hz, 2H), 5.06 (s, 1H), 4.93 (s, 1H), 4.68-4.60 (m, 1H), 4.47 (td, J=8.1, 5.4 Hz, 1H), 4.34 (dd, J=8.4, 3.9 Hz, 1H), 4.20 (dd, J=8.1, 3.9 Hz, 1H), 4.06-3.95 (m, 2H), 3.73 (bd, J=5.2 Hz, 1H), 3.63 (dd, J=16.7, 6.0 Hz, 1H), 3.54 (dd, J=16.8, 5.6 Hz, 1H), 3.03 (dd, J=14.1, 5.1 Hz, 1H), 2.95 (dd, J=14.2, 3.2 Hz, 1H), 2.81 (dd, J=13.9, 8.8 Hz, 1H), 2.65 (dd, J=14.2, 10.5 Hz, 1H), 2.34-2.29 (m, 2H), 1.91 (dd, J=14.7, 7.9 Hz, 2H), 0.99 (overlapped d, 6H); HRMS m/z calcd for C33H46N7O11 [MH+] 716.3255. found 716.3186; m/z calcd for C33H45N7NaO11 [MNa+] 738.3075. found 738.2970.Ie. Synthesis of a Genuine Sample of 14.
Fmoc-Thr(tBu)-NHBn (S30). Benzylamine (0.270 g, 2.52 mmol) was coupled to Fmoc-Thr(tBu)-OH(S29, 1.00 g, 2.52 mmol) using the standard HATU coupling procedure. The reaction was diluted with Et2O (150 mL), and washed with 10% citric acid solution (60 mL), water (3×60 mL), and brine (60 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified using flash chromatography (15% EtOAc/hexanes) to afford S30 as a white solid (1.20 g, 98% yield). Rf 0.53 (1:1 EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J=7.5 Hz, 2H), 7.61 (d, J=7.4 Hz, 2H), 7.44-7.27 (m, 9H), 6.06 (d, J=4.6 Hz, 2H), 4.50 (dd, J=7.8, 5.7 Hz, 2H), 4.44-4.37 (m, 2H), 4.27-4.14 (m, 3H), 1.24 (s, 9H), 1.04 (d, J=6.5 Hz, 3H); HRMS m/z calcd for C30H34N2NaO4 [MNa+] 509.2416. found 509.2226.
Fmoc-Phe-Thr(tBu)-NHBn (S31). S30 (0.300 g, 0.617 mmol) was subjected to the standard Fmoc deprotection procedure and then coupled to Fmoc-Phe-OH (0.263 g, 0.678 mmol) using the standard HATU coupling procedure. The reaction was diluted with Et2O (90 mL), and washed with 10% citric acid solution (40 mL), water (3×60 mL), and brine (60 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified using flash chromatography (30% EtOAc/hexanes) to afford S31 as a white solid (0.344 g, 88% yield over two steps). Rf 0.33 (1:1 EtOAc/hexanes); NMR (300 MHz, CDCl3) δ 7.77 (d, J=7.6 Hz, 2H), 7.53 (dd, J=7.1, 5.2 Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.34-7.10 (14H), 5.30 (d, J=6.2 Hz, 1H), 4.55-4.34 (m, 4H), 4.30-4.08 (m, 4H), 3.11 (d, J=6.4 Hz, 2H), 1.18 (s, 9H), 0.94 (d, J=6.1 Hz, 3H); HRMS m/z calcd for C39H43N3NaO5 [MNa+] 656.3100. found 656.2769.
Ac-Phe-Thr(tBu)-NHBn (S32). S31 (0.186 g, 0.293 mmol) was subjected to the standard Fmoc deprotection procedure. The resulting residue was dissolved in a solution of DCM (2 mL), Ac2O (0.060 g, 0.587 mmol), and DIEA (0.076 g, 0.587 mmol) and stirred for 30 minutes at rt. The reaction was diluted with EtOAc (60 mL), washed with water (20 mL), and brine (20 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation. The crude product was purified using flash chromatography (5% MeOH/DCM) to afford S32 as a white solid (0.115 g, 95% yield over two steps). Rf 0.36 (5% MeOH/DCM); 1H NMR (300 MHz, CDCl3) δ 7.34-7.13 (10H), 6.88 (d, J=5.8 Hz, 1H), 6.15 (d, J=7.3 Hz, 1H), 4.72 (q, J=6.8 Hz, 1H), 4.51-4.33 (m, 3H), 4.25-4.14 (m, 2H), 3.08 (dd, J=6.7, 4.2 Hz, 2H), 1.97 (s, 3H), 1.16 (s, 9H), 0.96 (d, J=6.3 Hz, 3H); HRMS m/z calcd for C26H36N3O4 [MH+] 454.2706. found 454.2490; m/z calcd for C26H35N3NaO4 [MNa+] 476.2525. found 476.2502.
Ac-Phe-Thr-NHBn (14). S32 (0.070 g, 0.154 mmol) was dissolved in an ice cold solution of TFA (0.600 mL)+DCM (0.150 mL)+Et3SiH (0.250 mL) and stirred for 0.5 h. The ice bath was removed and the reaction was allowed to stir at rt for an additional 2 hours. The reaction was diluted with EtOAc (40 mL), and washed with sat. NaHCO3 (2×25 mL), water (25 mL), and brine (25 mL). The organic layer was dried (MgSO4), filtered, and concentrated by rotary evaporation to afford 14 as a white solid (0.050 g, 82% yield). Rf 0.14 (10% iPrOH/DCM); 1H NMR (300 MHz, CD3OD) δ 7.33-7.15 (10H), 4.68 (dd, J=9.0, 5.9 Hz, 1H), 4.36 (s, 2H), 4.30 (d, J=3.7 Hz, 1H), 4.21 (dd, J=6.4, 3.7 Hz, 1H), 3.14 (dd, J=13.9, 5.9 Hz, 1H), 2.91 (dd, J=13.9, 9.0 Hz, 1H), 1.89 (s, 3H), 1.14 (d, J=6.4 Hz, 3H). The spectra obtained from this experiment overlaps perfectly with that of 14 prepared from the ligation/ring-opening procedure.